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Hi,
This is a follow up to my earlier question about low cMax values after
an IV bolus injection. I am intrigued by the possibility that this may
be the result of binding of my compound to RBCs. First, I would like to
get some idea of whether or not this is happening by doing an in vitro
experiment. Can I spike compound into freshly isolated mouse blood and
then extract the compound from either whole blood or plasma isolated
from this whole blood to get an idea of whether the compound is being
"trapped" in the RBCs? If so, can someone recommend a method for
isolation of compounds from whole blood? I have been using acetonitrile
precipitation followed by solid phase extraction for my plasma samples
and if there is an analogous protocol for whole blood (i.e. something
involving acetonitrile precipitation after a step to lyse the red cells
- freezing? I don't have access to a sonicator), that would be great.
Second, what are the implications for a compound highly bound by RBCs?
Is this compound ever available again to make its way to the target
tissue (tumor in my case)?
Third, what characteristics predispose a compound to be highly bound by
RBC. In the archives I read something about pKa values and ionization
states being important. Could someone please explain this?
Thanks again so much,
Noelle
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Spike the test article into blood and then analyze the blood as well as
blood processed into plasma. The same extraction method can be used for
blood versus plasma in most cases
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I don't think that you need a sonicator. I use freezing followed by
thawing
and perchloric acid precipitation for methotrexate in RBC and it seems
to
work fine. There are also lysing solutions (can't recall the "recipe"
but
should be very easy to find) for RBC that work faster than freezing (and
even water lysis might just work too...)
As for spiking the compound into mouse blood, you might want to
incubate the
mix at 37=BA for enough time (1 hr?).
Facundo Garcia Bournissen.
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Hello,
if your drug and metabolites are stable in acid, the easiest way is an
acid
precipitation of the proteins/RBCs, e.g. by 1M HClO4. However, you have
to
be careful with acid precipitation of whole blood samples, if your drug
is
easily oxidable: The oxygen bound to hemoglobin is known to yield
reactive
oxygen species during acid precipitation which might produce artefactual
metabolites (cf. eg. Anal. Biochem. 191,347 (1990); Biol Chem Hoppe
Seyler
371, 881 (1990)).
If your drug is highly lipophilic you have to use e.g. alcohols for
plasma
extraction and Tsuchihashi reagent for whole blood samples ( icecold
water
added to blood sample (1:1) and precipitated with mixture of ethanol /
chloroform (63/37, v/v, icecold), thoroughly vortexed and centrifuged at
12000 rpm for 5 min ). However, recovery is mostly a big problem with
lipophilic drugs which can be hardly solved.
Hope that helps.
Regards
Dieter
Dr. Dieter Gallemann
Merck KGaA
Institute of Drug Metabolism and Pharmacokinetics
Am Feld 32
D-85567 Grafing
Germany
Tel.: +49 (0) 8092-7008-11
Fax: +49 (0) 8092-7008-7011
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August 31, 2004: Noelle: binding of drug to erythrocytes.
Whole blood samples can be spiked with drug in much the same way as
plasma. In order to release the drug from the erythrocytes (in the
absence of sonication) you could add salt/water to the individual
samples prior to freeze-thawing the tubes in a dry ice-solvent bath.
The sample preparation for the assay of drug in pharmacokinetic lysed
whole blood samples can be then be continued by (a) solvent extraction
followed by separation of the organic solvent (b) reduction in solvent
volume (c) subsequent reconstitution of the dried extract in mobile
phase.
The solvent extraction approach is ostensibly more labor intensive, but
will provide the "cleanest samples" for assay and subsequently
reliable pharmacokinetic profiles.
The alternative approach of protein precipitation by ~6% perchloric
acid or acetonitrile can be useful at high concentrations of drug, but
has to be evaluated by experiment on an individual case basis. There
is more of a potentially interfering biological matrix with such
extraction/precipitation approaches.
Regarding partitioning studies in whole blood and handling you may
find the publication below useful.
McLean AM, Cefali EA, Roden JS, Gonzalez MA, Bialer M., "Stability of
diltiazem in different biological fluids." Biopharm Drug Dispos. 1991
Jul;12(5):327-34.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?
cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=1878529
Regarding characteristics, which predispose a compound to be highly
bound by RBC: There are examples of diuretic drugs, which bind to
carbonic anydrase inhibitors such as tripamide/indapamide, that
partition into the erythrocytes. It may well be that the erythrocytes
are rich in carbonic anhydrase enzyme and this provides the driving
force for the partitioning
Hope above helps,
Angus McLean
8125 Langport Terrace,
Suite 100,
Gaithersburg,
MD 20877
Tel 301-869-1009
Fax 301-869-5737
BioPharm Global
(http://home.comcast.net/~angusmdmclean/BGWEBSITE/home.html)
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)